In article <3uidfs$4cs at mserv1.dl.ac.uk>, p_rudolph at MORPH.SPACENET.DE (Peter
Rudolph) wrote:
> recently a coworker of mine ran into a problem:
> She created a library with PCR using randomized primers,
> then she digested the fragment and want to ligate it into a vector.
> The complete PCR fragment is 96bp, after digestion she gets
> two fragments: 29 and 67bp. She separated them by gel
> electrophoresis on a 2.5% AG.
> The problem is now: How to purify the bigger fragment from the
> gel slice with a really *high* yield?
> She tried freeze-and-squeeze and Qiagen-gel-ex but got
> only very low yields.
> Any suggestions?
> Peter.
Hi Peter and Co-worker:
We assume you have used QIAGEN's QIAEX Gel Extraction Kit. You've got low
yield from using QIAEX because of the following possible causes:
- Binding conditions: When binding the DNA to the QIAEX suspension, the pH
of the mixture of QX1 binding buffer + your sample + QIAEX suspension
should
be less than 7.5. To be safe we even prefer it to be below 7.0. Re-using
electrophoreses buffer can cause the pH to be too high--beyond the
buffering
capacity of QX1. You can simply checkthe mixture with a pH strip. If the
pH
is too high, add 10 ul of 3M NaAcetate pH 5.0 to the mixture.
- For agarose gel >2%, such as in your case, make sure you double the
amount
of QX1 binding buffer.
- Overdrying of QIAEX suspension during evaporation of ethanol containing
QX3
Buffer. Overdrying causes the DNA to bind too strongly to QIAEX particle.
Dry the pellet until you see a white rim around the edge of the pellet.
This
step is one of the reasons why we upgraded the QIAEX to QIAEX II, which
also
provide higher yield.
- Elution conditions: contrary to binding conditions, pH of elution buffer
should be greater than 7.5. We recommend 10mM Tris-HCl pH 8.5. If youelute
with water, be cautious because water pHsometime is acidic, although
theoretically it should be neutral.
QIAEX was designed to sufficiently recover fragments from 40bp to
50kb. Nevertherless, less than 50% of fragments smaller than 40 bp will
be recovered.
If you have any questions, please contact our friendly molecular biology
specialists in Technical Service dept. at 800-DNA-PREP (or 800-426-8157).
Thank you for your support of our products
QIAGEN Inc.
--
QIAGEN 800-362-7737
Temporary Email Address qiagen at kaiwan.com